We are studying how newly-made non-cytoplasmic proteins are sorted to their many distinct locations within cells. This involves protein assembly into membranes and selective protein transfer across membranes (secretion). Such non-cytoplasmic proteins are often made with amino-terminal leader sequences; we have purified the first membrane protein precursor with an intact leader sequence (M13 procoat) and the first "leader peptidase" which removes the leader. Our reconstitution of assembly with these pure components will allow us to study at a chemical level how polar regions of polypeptide cross the bilayer. The structure, biogenesis, and specificity of the leader peptidase, as well as the fate of the leader peptide, will be examined. Assays for new elements which may function in assembly will be developed. Mutants in leader peptidase which are being isolated in our lab will allow a determination of the spatial distribution of assembly intermediates.